During FY09, we focussed mainly on three subprojects. (1) Capsid assembly and polymorphism of retroviruses and their implications for infectivity. Retroviruses capsids are unusual in that they are assembled inside the maturing virion, not in the cytoplasm or the nucleus of the infected cell. The capsid protein is incorporated into the provirion as part of the Gag polyprotein which forms a spherical shell lining the membrane of the provirion. After the provirion has budded off, the maturational protease is activated and dissects Gag into its matrix (MA), capsid (CA) and nucleocapsid (NC) moieties. Protease inhibitors were the first antiviral drugs to be used successfully against HIV. Of the Gag fragments, CA reassembles to form the shell of the virus core, housing the viral RNA and replication enzymes. Evidence suggests that a correctly formed core is essential for infectivity;however cores are highly polymorphic. In FY08 we published our study in which cryo-electron tomography was used to visualize mature virions of Rous Sarcoma Virus, the prototypic alpha-retrovirus. The virions ranged from 105 to 175 nm in diameter and their cores were highly polymorphic. We observed angular cores in the form of irregular polyhedra;cores with continuous curvature including a few fullerene cones;and tubular cores. From the tomograms we estimated the number of CA subunits per assembled capsid and, from the virion diameters, their original complements of Gag. We found that RSV virions, like HIV, contain unassembled CA subunits and the fraction of CA that is assembled correlates with core type. These observations implied that initiation of capsid assembly is a critical determinant of core morphology. In FY09, we carried this investigation further by similarly analyzing a conditionally lethal mutant affected in capsid assembly. The primary mutation is a substitution in the major homology region (MHR), a 20-residue tract at the start of the C-terminal domain (CTD) of CA protein. A search for second-site suppressors identified a mutation in the N-terminal domain (NTD), that restores infectivity despite rendering the virus temperature-sensitive. In mutant virions produced at the non-permissive temperature, capsid assembly is obviated. At the permissive temperature, capsid assembly is restored, albeit with an altered range of polymorphism. Fewer tubular capsidsare assembled than in wild-type and more virions have closed irregular polyhedral capsids, and these are 30% larger than in wild-type. Gag processing appears to proceed normally. It follows that FY/AV-CA assembles more efficiently in situ than wild-type and has a lower critical concentration, reflecting altered nucleation properties. These observations are consistent with and further illuminate reported in vitro assembly properties of the purified proteins. (2) The structures of pentamers and hexamers of RSV capsid protein. Mounting evidence supports the view that retroviral capsids have fullerene closues of a hexagonal lattice of CA protein, whereby each capsid contains 12 pentamers, variously distributed, plus much larger and variable numbers of hexamers. According to the fullerene conjecture, a capsids shape depends on how the pentamers are distributed and it size on the number of hexamers. CA hexamers have been studied in planar and tubular arrays but the predicted pentamers have not been observed. We performed cryo-electron microscopic analyses of two in vitro-assembled capsids of Rous sarcoma virus. Both were found to be icosahedrally symmetric: one is composed of 12 pentamers;the other, of 12 pentamers and 20 hexamers. Fitting of atomic models of the two CA domains into the reconstructions showed three distinct inter-subunit interactions. These observations show how pentamers are accommodated at vertices, support the inference that nucleation is a crucial morphologic determinant, and imply that electrostatic interactions govern the differential assembly of pentamers and hexamers. This study was completed and published in FY09. (3) PA-457 is an effective antiretroviral drug that appears to act by inhibiting proteolytic processing of the precursor Gag polyprotein. We have started to investigate its mode of action further by performing electron tomography on thin sections of freeze-substituted HIV-infected cells after PA-457 treatment. Untreated wildtype HIV-1 exhibits the canonical mature HIV-1 morphology, with a thin layer of density at the particle membrane and well-defined, often conical, core. HIV-1 lacking the viral protease (PR) forms immature particles with or without PA-457 treatment. The immature particle features a thick shell of Gag-related density, apposed to the viral membrane, and no defined features in the viral core. As other recent studies have described, we observe that the Gag lattice appears not to form a complete shell, instead only covering a majority portion of the viral membrane. The morphology of wild-type HIV-1 treated with PA-457 differs from either immature or mature particles. These particles feature a thin shell of density underneath a portion of the viral membrane. This shell is distinct from the immature Gag lattice, probabaly reflecting incomplete Gag cleavage. Interestingly, most particles also exhibit a small spheroid core, indicating that core assembly is not completely inhibited. A similar morphology is observed for CA5 particles, which carry mutations inactivating the PR cleavage site that PA-457 is thought to inhibit.